Evanescent wave background fluorescence/absorbance detection

ABSTRACT

It is proposed to increase the utility of intracellular fluorescence and absorbance measurements for control of fermentation and cell culture by correcting on-line for background fluorescence of the media, also to be able to measure the fluorescence or absorbance of the fluid media in the presence of suspended cells or particles. The measurements could be extended to measure the optical properties of other fluids which contain suspended particles. This invention uses the characteristics of the evanescent wave at the surface of an optic waveguide. The wave penetrates into the less dense medium only to about the depth of about 1/2 wavelength. The thickness of media swept by the evanescent wave is much less than the size of a cell, thereby essentially separating the fluorescence or absorbance of the media from that of the cells. Apparatus is disclosed for carrying out the methods taught herein, including the use of an optical fiber to generate the evanescent wave and the use of a flat plate waveguide to generate it. Apparatus that can read both bulk fluorescence and evanescent wave fluorescence employs a flat plate waveguide to generate the evanescent wave and fiber optics to create an alternating dual-beam approach to generating both sets of data.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to method and apparatus for the detectionand measurement of certain optical characteristics of biological cultureand fermentation media in a bioreactor or the like, and moreparticularly, to the measurement of the fluorescence and/or absorbanceof a continuous phase in the presence of a discontinuous phase.

2. Description of Related Art

Fluorescence of intracellular NADH or NADPH has been shown to be a goodindicator of the metabolic state of cells in culture, as well as servingas an indicator of the concentration of cells in the culture medium.Several papers have attested to the value of this kind of in situmeasurement for both microbial and yeast culture applications. See,e.g., W. B. Armiger et al., Analysis and Control of Fed-BatchFermentations Producing Escherichia coli Using Culture Fluorescence,Proceedings Biotech 84, Washington, D.C. 1984. Apparatus for performingthese measurements is available from BioChem Technology, Inc., Malvern,Pa., as the FluoroMeasure™ System.

From an economic point of view, it would be advantageous to use lowercost complex nutrients, such as molasses or corn steep liquor inindustrially significant cultures. These, however, introduce additionalbackground fluorescence. If the medium contains a fluorescent componentwhich does not change during the course of the culture, a backgroundcorrection can be made simply by subtracting the reading at time zerofrom all subsequent readings. In a case where the fluorescence of themedia changes due to use by the cell or in the case where the cellsproduce a competing fluorescence, it becomes more difficult to correcton-line for changes in background. This can be done for a batch cultureby taking serial samples, removing the cells and measuring thefluorescence of the medium. Even more difficult are backgroundcorrections in cases of continuous culture or where nutrients are addedstepwise during the culture.

For fluorescent media, it would be useful to be able to determine howthe fluorescence of the media is changing during the fermentation, suchthat a measurement can be made to the metabolic state of the cells andtheir growth rate. What is needed is a means of effecting the separationof the cell fluorescence from that of soluble materials.

Optical sensors for fermentations or tissue culture are capable ofgiving information on intracellular substances and conditions. Suchinformation would permit a finer control based on actual intracellularinformation rather than on the existing on-line sensors, temperature,pH, dissolved oxygen, off-gas analysis. It would lead to a betterscaleup and commercialization of products derived from recombinant DNAand cell fushion technologies. Optical sensors, at present, work bestwith media which do not interfere since there is not easy way to correctfor changes in optical background.

The early work with optical sensors for following intracellularmetabolism dates back to 1957, when Duysen and Amesz observed that thefluorescence of baker's yeast was similar to that of NADH and that thefluorescence of starved yeast could be enhanced by adding ethanol orglucose to the suspension. Later, Harrison and Chance built aninstrument capable of measuring culture fluorescence in situ and couldmonitor aerobic/anaerobic transitions in continuous culture. Using asimilar device, Humphrey and coworkers, and others, have shown that afluorometer placed on a fermentor could measure intracellular NADHchanges and might be useful for process control. Zabriskie and Humphreyshowed the linear relationship between the logarithm of the fluorescenceof the culture and the logarithm of cell concentration. Ristroph et al.studied the relationship between culture fluorescence and the growth ofCandida utilis in a fed batch fermentation.

These studies have shown that the concentration in intracellular NADHmeasured by culture fluorescence in a fermentation is a function of thenumber of cells, the energy level within each cell, and the level ofmetabolic activity. A mathematical expression which is derived fromthese studies is:

    F(t)=[Y.sub.f/x (1+m(t))]X(t)+E(t)

X(t) is the cell concentration. The term in square brackets is thefluorescence yield, which is made up of an invariant compound Y_(f/x),which is characteristic of the type of organism and a variable componentm(t), which changes in response to shifts in the level of metabolicactivity. The final term, E(t), with which the present invention ismainly concerned, is the environmental, or background, fluorescence.Obviously, if E(t) fluctuates during the fermentation, then it would bedifficult if not possible to derive information about the cells from themeasured overall fluorescence. Continuous, or batch fed fermentation orcell cultures only exacerbate the problem. In those techniques,additional variables are introduced without corresponding information asto concentration.

Almost all of the published studies have used synthetic media where E(t)is low, or the corrections for E(t) had to be arrived at empirically, Inscaling up fermentations and cell cultures for commercial production,economic factors may dictate use of the natural nutrients, like molassesor fetal calf serum, which have a natural fluorescence and thereforecontribute to the background value. When checking some of theassumptions used in correcting for the background, I found indicationsthat the background fluorescence of, for instance, molasses, and thefluoresence of yeast cells do not add linearly. This pointed up the needfor a method for continuously measuring the media fluorescencebackground on-line and in real time, i.e. using a sensor or sensorscontinuously monitoring the detected variable as the fermentation orculture is being conducted.

This means that, without physically separating the cells from the media,a method was needed which caused the media to fluorescence without, atthe same time, causing the cells to fluoresce. In accordance with thepresent invention, the evanescent wave phenomenon is used to meet thisneed.

SUMMARY OF THE INVENTION

When a beam of light is totally reflected from a non-mirrored interfacebetween two optically transparent media of different refractive indexes,an evanescent wave phenomenon, such as shown in FIG. 1, exists. Thelight beam 11 is totally reflected from this kind of surface, unlike amirrored surface, and behaves as though it penetrates for about half awavelength into the less dense medium 12, e.g. an aqueous medium.Reference numeral 15 identifies the portion of light beam 11 that is theevanescent wave in the less dense medium 15. Reference numeral 16identifies a measuring arrow showing a distance that is one wavelengthof the light beam 11.

FIGS. 1 and 2 schematically show that the reflected beam is slightlydisplaced from where it would be if reflected from a mirrored suface.This displacement has been shown experimentally, and it is one of theproofs of the existence of the evanescent wave. This part of the lightbeam has many characteristics of a standing wave parallel to thesurface. FIG. 3 shows how the intensity decreases with distance from thesurface.

In FIG. 3, N is the incident wave, R is the reflected wave θ is theangle of incidence (which is greater than θ_(c), the critical angle. Zis the distance axis in the rarer medium measured from the interfacewith the more dense medium. E_(o) is the initial magnitude of theelectric field component of the light at zero depth in the rarer medium.dp is the depth of penetration, defined as the distance required for theelectric field to fall to e⁻¹ of its value at the surface. The value ofdp is directly related to the wave length in the denser medium and isinversely proportional to the angle of incidence and top the ratio ofrefractive indexes of the two media. The greatest strength of theevanescent wave occurs at the surface, and it decreases exponentiallywith distance from the surface.

It can be absorbed by an appropriate colored material, and if thematerial is fluorescent, it can excite the material to fluoresce. At thewave length of interest, 340 nm, the volume in liters swept out by thisevanescent wave over a one square centimeter area would be 1.7×10⁻⁸liter. For a 200 μm diameter optical fiber 2.5 cm long, the swept volumewould be 1.3×10⁻¹¹ liter.

The present invention accomplishes this separation by using thecharacteristics of the evanescent wave which forms in the less densemedium when light is totally reflected from the interface between twooptically transparent substances of different refractive indexes. Itmakes use of my observation that it is unlikely that an intact cell willbe in the volume of fluid swept by the evanescent wave next to theoptical wavelengths since the wave penetrates approximately only 1/3 to1/2 of a wavelength into the aqueous layer. The present inventiontherefore contemplate measurement of the fluorescence of the mediumwithout interference from the intracellular fluorescence or fromfluorescence of particles in solution.

The same concept is also adapted to measuring the optical absorbance ofthe medium independent of cells and particulate material. This cansignificantly reduce the complexity of the computer programs needed todeconvolute the data and thereby make the control of fermentation andtissue culture easier to achieve.

At the usual concentration of cells in a bioreactor, it is unlikely, asI said, that a cell would be in this small volume of fluid at any giventime. Also, since, at an excitation wavelength of for example 280 nm,the evanescent wave only penetrates about 110 to 170 nm into the liquidphase, even if the cell is resting right on the surface of the opticalwave guide, very little of the cell volume (mostly the cell wall or cellmembrane), will interact with the evanescent wave. Thus, by limiting thevolume that can interact with the light wave to that within theevanescent wave, the present invention provides, in effect, a separationof the intracellular fluorescence or absorbance from the fluorescence orabsorbance of the media.

It is an object of this invention to facilitate the opening up offermentation and cell culture to intracellular optical measurement undera wider variety of culture conditions because there will be a way tocorrect continuously for environmental or background changes on-line inreal time.

It is a further object of the present invention to provide for theseparation of the optical effects of intracellular contents from theoptical effects of the culture medium.

It is a further object of the present invention to follow changes in theculture medium without interference from changes in the intracellularcontents.

It is a further object of the present invention to follow changes in theintracellular contents without interference from changes in the culturemedium.

It is a still further object of the present invention to reduce costs ofmonitoring the fluorescence of cell cultures.

It is a still further object of the present invention to simplify andshorten the time for scaleup or change of media in a fermentation, sinceseparate, offline empirical measurements of background or environmentalfluorescence will not have to be made. By a single test run, cultureconditions might be brought to a preliminary optimization by appropriateadditions of media components.

It is a still further object of the present invention to provideinformation about the extent of cell rupture, whether due to shearforces or other mechanical or chemical causes. In accordance with thepresent invention, the effect of stirring forces on cell integrity couldbe measured on-line in real time, allowing corrections to be made duringa fermentation run rather than after the run when data shall beensubsequently analyzed.

It is a still further object of the present invention to permit the useof optical absorbance methods for following other non-fluorescentintracellular materials, since the ability to correct for backgroundwould provide the equivalent of a continuous dual beamspectrophotometer.

It is a still further object of the present invention to provide for thebetter control of the concentration of individual nutrients in theculture media through the improved ability to follow specific changes ofoptically differentiable materials in the medium.

It is a still further object of the present invention to significantlyimprove the yield of fermentation and tissue cultures, reduce the timeand cost of scaleup, allow for a more precise control based on the stateof the intracellular metabolism, and speed up the commercialization ofnew recombinant DNA and cell fushion technologies through more efficientfermentation and tissue culture techniques.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of the evanescent wave phenomenon,wherein a light beam 11 traverses a path from left to right through awave guide 13 such as an optical fiber.

FIG. 2 is an enlarged view of the circular area 2 within FIG. 1.

FIG. 3 is a schematic illustration of the variation of intensity of theevanescent wave with distance from the interface between the two media.

FIG. 4 is a partially schematic illustration of a cross-sectionalelevation of one of the variations of the present invention employing ametal sheathed optical fiber that may be dipped into a liquid medium.

FIG. 5 is a cross sectional side elevation of the embodiment shown inFIG. 4 viewed from a right angle to the view of FIG. 4, taken along thecross sectional line 5--5.

FIG. 6 is a partially schematic illustration of another embodiment ofthe present invention.

FIG. 7 shows the portion of the embodiment of FIG. 6 viewed from a rightangle to the view of FIG. 6, taken along line 7--7.

FIG. 8 is a partially schematic illustration of a cross-sectionalelevation of a preferred embodiment of the present invention wherein theoptical waveguide is a flat plate 1316, and a barrier 1312 confines theliquid to one side of the waveguide.

FIG. 9 is an elevation view of the disk 1328 shown in FIG. 8 taken alongthe line 9.

FIG. 10 is a cross sectional side elevation of the embodiment shown inFIG. 8 viewed from a right angle to the view of FIG. 8, taken along thecross sectional line 10--10.

FIG. 11 is an enlarged view of the area 11 within FIG. 8.

FIG. 12 is a schematic illustration of the embodiment of FIG. 8 showingthe paths of light rays and the steps of processing of data.

FIG. 13 is a schematic illustration of the contents of the source anddetector housing of an embodiment alternative to the design shown housedin element 1331 of FIG. 8.

FIG. 14 shows a portion of the embodiment of FIG. 13 viewed from a rightangle to the view of FIG. 13 taken along the line 14.

DETAILED DESCRIPTION

The embodiment of the invention shown in FIGS. 4 and 5 provides arelatively simple means of determining the background fluorescence of asolution containing particulate matter by dipping into the liquid adipstick 40 which comprises a metal housing 41 surrounding an opticfiber 44 through which an evanescent wave of light is used to excite thefluorescence of the solution. The housing 41 defines a chamber 47, intowhich the solution to be tested passes through apertures 46 in thehousing 41.

On the portion of the optic fiber 44 within the chamber 47, the opaquesheath 42 and transparent cladding 43 have been removed, exposing thefiber 44 directly to the solution within the chamber 47.

To illuminate the fiber 44, there is provided a light source 1301 ofeither visible or invisible light, such as an incandescent lamp orlaser, an excitation beam lens 1302, and an excitation beam filter 1303,arranged as shown in FIG. 4.

The radiation from the light source 1301 passes through the lens 1302,where it is collimated, and then passes through the filter 1303, whereit is filtered into a monochromatic excitation beam 1326 of desiredwavelength (schematically illustrated by dashed lines with arrows) andpasses through an aperture in plate 1340.

The excitation beam 1326 thereupon encounters a dichroic mirror 1349adapted to reflect the wavelength of light represented by the excitationbeam 1326 into the excitation-emission beam lens 1358, where the beam1326 is decollimated and directed into proximal end of optic fiber 44,which may extend from the metal housing 41 if desired. As stated above,the excitation beam 1326 travels through optic fiber 44 until it reachesthe distal end, where it encounters a light trap 45, e.g. of blacksilicone rubber.

The evanescent wave portion of the excitation beam 1326 traveling downthe optic fiber 44 encounters the molecules of solute within chamber 47that are immediately adjacent the fiber 44 (i.e. within about 1/3 to 1/2wavelength as discussed above) and, to the extent that it issusceptible, excites the solute to emit fluorescence.

Such fluorescence passes into the optic fiber 44 and travels to itsproximal end as the emission beam 1327, where it exits the optic fiber44, encounters the excitation-emission beam lens 1358 and is collimated.The beam 1372 then passes through the dichroic mirror 1349, which hasbeen fashioned to pass the wavelength of the light emitted by thefluorescence of the solute to be measured.

The emission beam then impinges upon an electronic detection system 1323such as a photomultiplier tube in counting mode. The detection system1323 is selectively responsive to the wavelength of the emissionradiation because a filter or monochromator is incorporated therein. Theelectronic detection system 1323 generates a signal that is sent to alock-in ratio amplifier 1344. A reference detector 1350, which detectsthe intensity of the excitation beam 1326, also generates a signal thatis sent to the lock-in ratio amplifier 1344, where the two signals areprocessed conventionally to compensate for variations in the intensityof the excitation beam 1326.

The signal from the lock-in ratio amplifier 1344 is transmitted to ananalog-to-digital converter 1345, which generates a signal fed to adigital display panel or signal processor 1346. Desirably an additionaldigital signal 1352 from a conventional device reading bulk fluorescence(of the solution and any particulate matter in it) is generated andsimilarly fed to the display panel or processor 1346. The signal 1352may, for example, be from a Fluoromeasure™ fluorometer (BioChemTechnologies, Inc., Malvern, Pa.). When the data in signal 1345 issubtracted from the data in signal 1352, the resulting data sent toelement 1351 describes the fluorescence of the particulate matter,inasmuch as fluctuations in the fluorescence of the solute have beensubtracted from the fluorescence of the bulk.

An alternative embodiment shown in FIGS. 6 and 7 utilizes an optic fiber62 within a housing 61 defining a chamber 67 having a pair of ports 72,73. The slurry to be subjected to optical measurement in accordance withthe present invention may be introduced through inlet port 72 andexhausted through outlet port 73. Desirably the chamber 67 is designedso that flow therethrough is essentially laminar.

As in the embodiment previously described (FIGS. 4 and 5), light from alight source 1301 passing through a lens 1302 and filter 1303 isintroduced into the end of the fiber optic 62. However, to maximize theevanescent wave relative to the radiation traveling straight through thefiber optic 62, a plate 1340A, having an O-shaped aperture, with afilled-in center, may be used rather than one having a circular cutout.This-will block rays from entering the fiber optic 62 along the axis.

In this configuration, a reference detector 1350 is disposed along adifferent path from the light source 1301 than that traveled by theexcitation beam 1326 so that variations in the intensity of the lightsource can be detected and fed to a lock-in ratio amplifier 1344 as isconventional.

At the distal end of the fiber optic 62, an electronic detection system1323 is placed to receive light therethrough. The detection system 1323may be set up to detect the intensity of light of the wavelength of theemission beam 1326. In that event, it will generate a signal useful indetermining the absorbance of the solute in solution, free of cellularor other particulate matter. The absorbance information may be relatedto the concentration of a solute that is to be monitored, or it may be abackground figure which may appropriately be subtracted from anotherabsorbance reading to provide useful data.

Alternatively the detection system 1323 may be set up with anappropriate filter or monochromator to detect a wavelength of radiationwhich is emitted by a solute as fluorescence, in which event theresulting data will be similar to that generated by the embodiment ofthe invention shown in FIGS. 4 and 5.

Similarly to the previously described embodiment, the signal fromdetection system 1323 is supplied to the lock-in ratio amplifier 1344,and the output thereof is directed to a display panel or processor 1346,the output of which may, for example, be fed to a chart recorder 1353 asshown.

Using the evanescent wave phenomenon, the embodiment of the presentinvention shown in FIGS. 8 to 11 is capable of determining bothabsorbance and fluorescence in such quick alternating succession as toprovide virtually simultaneous readings. With a flat plate 1316 as thewave guide, the device housed in detector enclosure 1332 is particularlyadapted to be used in a reactor or fermentation vessel 1312 for realtimedetermination of several variables which assist in determining theinstantaneous concentration of various components of the contents of thevessel 1312.

The detector enclosure 1332 is mounted within a conventional pipelikemounting port 1362 extending from the reactor vessel wall 1312. Thedistal end of mounting port 1362 is threaded to mate with grommet 1333,which secures the enclosure 1332 to the port 1362 and thereby to thereactor vessel wall 1312.

Depending on the length of the mounting port 1362 and the depth to whichit is desired that the detector housing 1332 penetrate beyond the vesselwall 1312 into the reaction mixture 1314, a rubber-like O-ring 1311 isinterposed within any of three O-ring grooves 1361 to seal the retainersleeve 1354 of housing 1332 watertight within the port 1362.

A screw-threaded wave guide plate mounting sleeve 1336 mates with theretainer sleeve 1354 and holds the wave guide plate 1316 securely inplace. An insert 1335, which may be one of optionally several lengths,extends the wave guide plate mounting sleeve 1336 so that the wave guideplate 1316 extends the desired distance into the reaction mixture 1314beyond vessel wall 1312.

A fluorescence enclosure 1308 extends outwardly from the vessel wall1312, screw-threaded to the insert 1335. Enclosed within the aforesaidelements are optical fiber elements 1305, 1317, 1319 and 1320, whichconvey light to and from the wave guide plate 1316. The optical fiberelements 1305, 1217, 1319 and 1320 pass through fluorescence enclosurecover 1307, which is held in place by a fluorescence enclosure coverretainer bezel 1309.

The light source 1301, lens 1302, filter 1303 and aperture 1340 aregenerally as have been described above. As shown schematically in FIG.12 as well as generally in FIG. 8, an excitation beam light chopper 1325is interposed in the optical path to pass the focused excitation beam1326 to the evanescent wave excitation fiber optic 1305 and then to thedirect wave excitation fiber optic 1320 in alternating succession. Asshown more particularly in FIG. 9 the excitation beam light chopper 1325comprises a disk 1328 having mounted thereon a semicircular mirror 1329,the other half of the disk 1328 having an opening 1342 sufficiently wideto allow the excitation beam 1326 to pass through to the optical fiber1320.

The chopper disk 1328 is rotated by a shaft 1330. As the disk 1328rotates, the excitation beam 1326 directed into two alternate paths.When the excitation beam 1326 is incident on the mirror 1329, the beamfollows path 1326A directed to optic fiber 1305. Alternately, when thedisk 1328 has rotated to a position where the excitation beam 1326 isincident on the opening 1342, it passes through to the optical fiber1320.

The optical fiber 1305 carries the excitation beam 1326A from thesealed, light-tight housing 1331, to fluorescence detector enclosure1332. The optical fiber 1305 typically consists of several individualfibers completely surrounded by a flexible transparent cladding 1306 andan opaque flexible sheath 1304. The refractive index of the transparentcladding 1306 is slightly less than that of the optic fiber 1305.

After passing through grommet 1333, the individual optical fibers of theoptic fiber 1305 pass through a mounting block 1355 where they arespread out, as shown in FIG. 10. Optic fiber mounting block 1355 isdesirably injection molded of a plastic capable of withstandingsterilization temperature of about 140° C., e.g. polysulfone.

The individual fibers of optical fiber 1305 that are in contact with theprism 1310 along its oblique surface are cut square to the longitudinalaxis of the fibers. The mounting block 1355 holds the fiber bundle 1305such that the light enters at right angles to the oblique surface of theprism 1310. The angle of the oblique surface of prism 1310 to the sideof the prism in contact with the flat plate wave guide 1316 is such asto introduce the light beam 1326A into the flat plate wave guide 1316 atan angle greater than the critical angle, so that the light beam 1326Awill be confined to the flat plate wave guide and will generate anevanescent wave at the interface of the wave guide 1316 and the reactionmedium 1314 in contact with the wave guide.

Prims 1310 is rectangular and has the same refractive index as the flatplate wave guide 1316. The loss of intensity of excitation beam 1326during its transition from the optic fiber 1305 to the prism 1310 isminimized by having the sides of the prism 1310 greater than thediameter of the optical fiber 1305 and by having the transitionalinterface between the optic fiber 1305 and the slanted surface of theprims 1310 covered by a liquid 1324 having same refractive index as thatof the prism 1310.

The flat transparent side of the prism 1310 is cemented to the distalface 1337 of the flat plate wave guide 1316 by utilizing a transparentcement having a same refractive index as that of prism 1310 and flatplate wave guide 1316. The actual angle of the oblique surface of prims1310 is a function of the refractive indices of the materials used forprism 1310 and the flat plate wave guide 1316. The flat plate wave guide1316 is circular in cross section and is typically made of bubble-freeand distortion-free material such as quartz. The two parallel faces 1337and 1338 are optically polished to a high degree and are truly parallelwithin the normal manufacturing tolerances. The cylindrical side wall ofthe flat plate wave guide 1316 is significantly less in height than itsdiameter.

The flat plate wave guide 1316 is sealed along its side wall by anopaque seal 1356 of PTFE polymer (e.g. Teflon or the like) to preventloss of light and also act as a sealent. The frontal side 1338 of theflat plate wave guide 1316 is coated with a very thin and hardtransparent layer 1315 of material such as Surlyn (Dupont), depositeddiamond etc. The thickness of the wave guide coating 1315 should be suchas to have no effect on the penetration of the evanescent wave 1339 intothe medium 1314. The wave guide coating 1315 is desirable to prevent theadherence of cellular products generated by the particulate matter 1313or the dirt present in the medium 1314. The wave guide coating 1315 alsoprevents damage such as scratches to the frontal side 1338 of the flatplate wave guide 1316.

An evanescent wave 1339 is created within the flat plate wave guide 1316when the excitation beam 1326 is repeatedly reflected between the twonon-mirrored surfaces 1337 and 1338 respectively. The evanescent wave1339 so generated penetrates into the medium 1314 under observationthrough the frontal side 1338 of the flat plate wave guide 1316. Asexplained above, the evanescent wave only penetrates a distance up toabout half of a wave length of the excitation beam 1326 into the medium1314 under observation. The discrete particulate matter 1313 such ascells present in the medium 1314 has virtually no interaction with theevanescent wave 1339.

There is a prism arrangement 1342 similar to prism 1310 at the oppositeend of the flat plate wave guide 1316 along its distal side 1337 asshown in FIGS. 8 and 12. The thickness of the flat plate wave guide andthe distance between the prisms 1310, 13411 is such that the incidentlight beam 1326A is refracted an integral number of times and exitsthrough prism 1341. The ends of the individual fibers of fiber optic1317 have been cut square to the longitudinal axis of the fibers. Themounting block 1355 holds the fiber bundle 1317 such that itslongitudinal axis is at right angles to the oblique surface of prism1341.

As an alternate construction, the flat plate wave guide 1316 and prims1310, 1341 may be fabricated as a single unit. Moreover, alternativelyto the relationship illustrated herein, wherein the faces of the prims1310, 1341 are raised above the surface of the wave guide 1316, theoblique faces of the prisms may be recessed into the surface of the waveguide. As an additional alternative, two diametrically opposite edges ofthe wave guide plate may be beveled to serve an equivalent function tothe oblique edges of prisms 1310 and 1340.

Individual fibers of the optical fiber 1317 are attached to an obliquesurface of the prism 1341, the transitional interface consisting of aliquid film 1324 having a refractive index close to that of the flatplate wave guide 1316. The optical fiber 1317 then passes through theoptic fiber mounting block 1355 and its cross section then becomingcircular. The optical fiber 1317 then passes through the grommet 1333and enters the source and detection housing 1331. In direct path ofoptical fiber 1317 as shown in FIG. 8 there exists a light chopperassembly 1318 which is constructed similarly to excitation light chopper1325. Next to the light chopper assembly 1318 in the same directionthere is an emission beam filter 1312 which eliminates all thenonfluorescent light, followed by the emission beam lens 1322 whichfocusses the emission beam 1327 on an electronic detection system 1323.

FIG. 12 is a schematic representation of the light path in theembodiment of the present invention shown in FIG. 8. The light beam 1326from light source 1301 is focused by a lens 1302 such that the light isproperly coupled to the optical fibers bundles 1305 and 1320. Incoupling light to the optical fibers or wave guides, attention must bepaid to the numerical aperture ("NA") of the fiber optic or wave guide.It is a matter of matching the lens 1302 to the NA and the diameter ofthe fiber or fiber bundle or quartz wave guide.

Care must be taken to prevent the fiber cladding from acting as a waveguide. This can be achieved by sheathing the cladding with an opaquesheath. To get uniform distribution of light, the lens must confine thelight uniformly across the input face of the fiber or wave guide. Thelight then passes through a slit or diaphragm and thence to chopper1325.

Chopper 1325 then, by alternately interposing and removing the mirror1329 from the excitation light beam 1326, divides the light beam 1326into two paths, 1326A and 1326B.

Beam 1326A travels through fiber bundle 1305 to prism 1310, whichcouples the light beam properly to flat plate wave guide 1316 such thatthe light beam is guided by multiple internal reflection through thewave guide. The evanescent wave is absorbed by the solution componentsable to interact with light of the selected wavelength. The evanescentwave does not optically interact with or excite to fluorescence theparticles 1313 suspended in the reaction medium 1314. Those solutioncomponents able to fluoresce will emit their fluorescence at or near thesurface of the flat plate wave guide 1316.

A portion of the emitted light will couple into the wave guide and betransmitted through light path 1327A to chopper 1318. Light beam 1326Bis transmitted via fiber bundle 1320 to the internal surface 1337 of theflat plate wave guide, which acts as a window; since the light impingesat right angle to the surface, it goes right through and illuminates thebulk suspension near the flat plate 1316, thus exciting to fluorescenceboth the solution 1314 and the particles or cells 1313 suspended thereinwhich are able to fluoresce.

A portion of the emitted fluorescence beam 1327B passes back through theflat plate 1316 and enters fiber bundle 1319 and is guided to chopper1318. Chopper 1318 is synchronized with chopper 1325 throughsynchronizer 1348 such that when chopper 1325 is diverting the lightbeam over pathway 1326A, chopper 1318 is configured to allow lightthrough pathway 1327A to go through filter 1321 and lens 1322 todetector 1323. When chopper 1325 is diverting the light beam overpathway 1326B, chopper 1318 is configured to allow light through pathway1327B to go through filter 1321 and lens 1322 to detector 1323.

Synchronizer 1348 also serves to synchronize the signal processing trainwith the chopper positions such that the signal processor is is treatingthe signal as is appropriate to the mode of generation of the signal,e.g. determination of evanescent wave fluorescence vis-a-visdetermination of bulk fluorescence.

The signal from the detector 1323 and from synchronizer 1348 is fed, forexample, to an AC to DC converter and linearizer 1343, then to a lock-inratio amplifier 1344, where the amplitude of the signal is corrected forvariations in the amplitude of the excitation beam detected by referencedetector 1350, then to an analog-to-digital converter 1345, then to adigital display or signal processor 1345 and then to a digital storageor memory 1347.

If filter 1321 is constructed to pass the emitted fluorescencewavelength, then the device of the present invention measuresfluorescence. If filter 1321 is constructed to pass the same wavelengthas the excitation beam 1326, then the device measures the opticalabsorbance of the solution 1314.

In the event that it is desired that only fluorescence and not opticalabsorbance be measured, an alternative embodiment (not shown) may omitthe chopper 1318 of FIGS. 13 and 16. In that event, light beam 1327A ismerely trapped rather than being guided to filter 1321 and detector1323. Fiber optic 1317, in such an embodiment, may be omitted andreplaced with a light trap, or fiber optic 1317 may itself channel thelight away from the flat plate wave guide 1316 as a light trap. Both thebulk fluorescence excited by light beam 1326B and the solutionfluorescence excited by light beam 1326A generating an evanescent waveat the surface of the flat plate wave gude 1316 are transmitted overlight path 1327B to filter 1321. Filter 1321 is selected to allow onlythe emitted fluorescent wavelength to pass through.

FIG. 13 illustrates yet another embodiment where the contents of housing1360 are substituted for the contents of housing 1331 of the embodimentof FIG. 8. This embodiment is suitable for slow speed chopping of thelight beams, for example, a few hertz or even fractions of a hertz. Thechopping device viewed in the direction of arrow 14 is shown in FIG. 14.A flat plate 182 is affixed to a shaft 181, which is attached to a speedreducer clutch 183. The clutch 183 is attached to the shaft ofreversible motor 184. A flat mirror 171 is attached to plate 182.

The plate 182 with mirror 171 attached is pivoted to swing between theposition shown in FIG. 13 with solid lines or alternately the positionshown with dashed lines. A stop 172 limits the travel of mirror 171 asthe plate 182 abuts it and acts as a rigid point fixing the position ofthe mirror precisely again and again. Mirror 173 is similarly mountedfor reciprocation and synchronized with mirror 171, generally asdescribed with respect to the embodiment of FIGS. 8 and 12.

Having thus described my invention, what it is desired to protect byLetters Patent and hereby claim is:
 1. A method for the optical analysisof a slurry, which slurry comprises:(a) a continuous phase whichcomprises a component that fluoresces at an optically detectablewavelength, and (b) a discontinuous phase which comprises a componentthat also fluoresces at said optically detectable wavelength,the methodcomprising the steps of: (i) optically exciting said slurry with anevanescent wave having a wavelength that excites the fluorescence ofsaid components and simultaneously (ii) detecting the intensity of thefluorescence resulting therefrom, and then (iii) relating said detectedintensity of fluorescence to the concentration of said component in saidcontinuous phase, such that said detected intensity of fluorescence isindependent of the concentration of the component in said discontinuousphase that also fluoresces.
 2. A method for the optical analysis of aslurry, which slurry comprises:(a) a continuous phase which comprises acomponent that fluoresces at an optically detectable wavelength, and (b)a discontinuous phase which comprises a component that also fluorescesat said optically detectable wavelength,the method comprising the stepsof: (i) optically exciting said slurry with an evanescent wave having awavelength that excites the fluorescence of said components andsimultaneously (ii) detecting the intensity of the fluorescenceresulting therefrom, and at a different but nearby time (iii)illuminating the slurry with a non-evanescent wave and simultaneously(iv) detecting the intensity of the fluorescence or absorbance resultingtherefrom, and then (v) determining the difference between the twointensity values, and (vi) relating said difference to the concentrationof said component in said discontinuous phase, such that said differenceis independent of the concentration of the component in said continuousphase that also fluoresces.
 3. The method of claim 2 wherein thecontinuous phase comprises an aqueous solution in a bioreactor and thediscontinuous phase comprises living cells.
 4. The method of claim 3wherein the fluorescence of NADH and NADPH are measured.
 5. The methodof claim 4 wherein the excitation beam is about 366 nm and thefluorescence is measured at about 460 nm.
 6. Apparatus for determiningthe fluorescence or absorbance of the discontinuous phase of a systemhaving a continuous phase which also fluoresces or absorbs light whenexcited or illuminated by light at an optically detectable wavelengthemitted by the apparatus, comprising:(a) means for exciting thecontinuous phase with an evanescent wave at said wavelength, and (b)means for measuring the intensity of the fluorescence or absorbanceresulting from the excitation of the continuous phase, and (c) means forilluminating the system with a nonevanescent wave at said wavelength,and (c) means for measuring the intensity of the fluorescence orabsorbance resulting from the illumination of the system, and (e) meansfor comparing an intensity measurement generated by one of the aforesaidmeasurement means with an intensity measurement generated by the otherof the aforesaid measurement means and for generating an indication ofthe difference between them,such that the value of said difference isrelated to the concentration of said component in said discontinuousphase, and such that said value is independent of the concentration ofthe component in said continuous phase that also fluoresces. 7.Apparatus of claim 6, further comprising:(f) means for deactivating saidevanescent wave excitation means (a) before activating the measurementmeans (d) and for deactivating said illumination means (c) beforeactivating the measurement means (b), such that measurement means (d) isactive only during the illumination of the system by illumination means(c), and measurement means (b) is active only during the excitation ofthe continuous phase with evanescent wave excitation means (a). 8.Apparatus of claim 7, further comprising:(g) a source of light at saidwavelength, (h) means for directing light from said source alternatelyto said excitation means (a) and to said illumination means (c), (i)means for generating a signal having a value which is related to theintensity of light at said wavelength that is detected, (j) means fordirecting light from the system to said signal-generating means (i), and(k) means for detecting when the light-directing means (h) is directinglight to said excitation means (a) and when it is directing light tosaid illumination means (c) and for alternately directing the signalfrom said signal-generating means (i) to said measuring means (b)whenever the light is directed to excitation means (a) and to saidmeasuring means (d) whenever the light is directed to illuminating means(d).
 9. Apparatus of claim 8, wherein the excitation means (a) comprisesan optical fiber at least a portion of which is not covered withcladding.
 10. Apparatus of claim 8, wherein the excitation means (a)comprises an optical plate.
 11. Apparatus for determining thefluorescence or absorbance of the discontinuous phase of a system havinga continuous phase which also fluoresces or absorbs light when excitedor illuminated by light at an optically detectable wavelength emitted bythe apparatus, comprising:(a) a source of light at said wavelength, anda plate waveguide having a face in contact with said system, (b) meansfor directing light from said source to said plate waveguide at an anglesufficiently oblique to said face to create an evanescent wave in saidsystem, such that a first state of excitation of the system is created,(c) means for directing light from said source to said plate waveguideat an angle approximately perpendicular to said face, such that a secondstate of excitation of the system is created, (d) chopper means foralternately directing light from said source to said directing means (b)or said directing means (c), (e) means for detecting light at awavelength emitted by the system upon fluorescence, (f) means fordirecting light from said plate waveguide at an angle approximatelyperpendicular to said face to said detection means (e), (g) means fordirecting light from said plate waveguide at an angle oblique to saidface to said detection means (e), (h) chopper means for alternatelydirecting light from said directing means (f) or said directing means(g) to said detection means (e), (i) means for synchronizing choppermeans (d) with chopper means (h), and (j) means for processing thesignal from detection means (e) to compare the value obtained during thefirst state of excitation with that obtained during the second state ofexcitation and to produce output related to the difference in saidvalues.
 12. Apparatus of claim 11 wherein said means for directing light(b), (c), (f) and (g) are fiber optics.
 13. Apparatus of claim 12,wherein said means for directing light (c) and (f) together comprise asingle bundle of optical fibers.